Dietary calcium supplementation has been recommended for prevention of osteoporosis and has become a standard component of most "health food" diets. Biliary calcium has been recognized to play a central role in the formation of pigment gallstones. We have recently demonstrated that 5 days of oral calcium supplementation significantly increases biliary calcium in the prairie dog (K. D. Lillemoe, T. H. Magnuson, G. E. Peoples, et al., Gastroenterology 94: A563, 1988). We hypothesized, therefore, that long-term oral calcium supplementation would promote pigment gallstone formation. Sixteen adult male prairie dogs were maintained on a standard nonlithogenic diet. Eight animals received calcium supplementation (2.5 x control levels) in their water, while the remaining eight animals served as controls. After 8 weeks, cholecystectomy was performed, and the common bile duct was cannulated. Bile was examined microscopically and analyzed for ionized calcium, bilirubin, glycoprotein, and biliary lipids. The cholesterol saturation index (CSI) was calculated. Pigment stones and calcium bilirubinate sludge were present in all animals receiving calcium supplementation. Only one control animal had evidence of pigment stones (P less than 0.001). Biochemical analysis of gallbladder bile demonstrated a significant increase in total bilirubin and bilirubin monoglucuronide (P less than 0.01) as well as bile glycoprotein content (P less than 0.05) after oral calcium supplementation. Gallbladder bile ionized calcium was also increased although not significantly. These data suggest that oral calcium supplementation promotes gallbladder sludge and pigment gallstone formation in the prairie dog. This observation raises concern that oral calcium supplementation, especially in the older female population, may enhance gallstone formation.